Project Summary/Abstract Bacterial drug resistance is a significant global health threat that has turned once treatable bacterial infections (including Gram-positive infections) into deadly illnesses. To overcome this serious threat to human health, completely novel antibiotics are needed. Noncoding ribonucleic acids regulate a wide array of important cellular processes and represent a novel set of targets for developing the next generation of therapeutic agents to treat infectious diseases. The T-box riboswitch is a noncoding RNA that regulates the expression of essential genes in many Gram- positive bacteria (including pathogenic examples), thus making it a unique target for novel antibacterial agents. A key component of the T-box riboswitch regulatory mechanism involves tRNA binding to the highly conserved antiterminator RNA element--an excellent target for drug discovery. This project will explore the rational design of allosteric modulators of T-box antiterminator RNA function and compare their efficacy to that of competitive inhibitors. No therapeutic agents are currently known to specifically target the T-box riboswitch. This proposal seeks to improve the activity of an initial set of compounds by investigating the hypothesis that leveraging allosteric modulation is an effective drug discovery strategy for targeting noncoding RNA function. Specific Aims are to 1) Design potent antagonists of T-box antiterminator function (using computational methods informed by experimental data), 2) Synthesize compounds and screen in 1 assays (assays for antiterminator and riboswitch function and preliminary antibacterial screening); and, 3) Characterize compound binding and mechanism of action (quantifying antiterminator binding, riboswitch inhibition and using in vitro and in cellular structure probing methods). Achievement of the proposed specific aims will improve scientific knowledge by developing design strategies to identify inhibitors (including allosteric modulators) of RNA function and by exploring the most effective chemical space features of these small molecules. This information will be significant for antibacterial drug discovery targeting the T-box riboswitch and also for the still emerging broader field of targeting noncoding regulatory RNA in general for antibacterial, antiviral, anticancer and other drug discovery efforts. In the process, a team of primarily undergraduate students will gain first-hand experience working on an innovative drug discovery project that will inspire them to consider careers in biomedical research.